Arabidopsis 2010: An Isotope-Assisted Quantitative Phosphoproteomics Approach to AtHK1-Mediated Osmosignaling in Arabidopsis thaliana

拟南芥 2010：同位素辅助定量磷酸化蛋白质组学方法研究拟南芥中 AtHK1 介导的渗透信号传导

• 批准号: 0929395
• 负责人: Michael Sussman
• 金额: $ 120万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-15 至 2014-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0929395&HistoricalAwards=false
• 关键词: Arabidopsis; 2010; Isotope; Assisted; Quantitative

INTELLECTUAL MERIT As sessile organisms plants have evolved multiple mechanisms to sense and respond to many different environmental inputs, including abiotic conditions (eg. light, water availability) and biotic factors (eg. insects, pathogens). This project will advance our understanding of plant response mechanisms by defining the specific molecular steps involved in transmitting environmental signals to plant cells. Using the model plant Arabidopsis thaliana and advanced techniques in mass spectrometry (isotope-assisted quantitative phosphoproteomics) this project will identify proteins that are modified by phosphorylation as part of important signaling pathways. The initial emphasis will be on AtHK1, a plasma membrane histidine kinase that acts as a receptor for sensing and responding to changes in water availability (drought) to plant cells. This protein is one of a family of several dozen histidine kinases that initiate short- and long-term responses to changes in plant hormones and environmental parameters. Histidine kinases act, at least in part, by a series of phosphotransfers between histidine and aspartyl residues within three different proteins. These modifications (phosphorylations) ultimately result in large-scale changes in transcription, metabolism, cell division and differentiation. In addition to histidine and aspartate phosphorylation, genetic studies of drought responses have implicated a calcium-dependent signaling cascade that includes a serine/threonine protein kinase. No clear model has emerged for integrating these chemically distinct protein phosphorylation systems in the overall response to water availability. In this project, quantitative phosphoproteome measurements via in planta metabolic labeling with non-radioactive heavy isotopes and tandem mass spectrometry will be used, together with mutants displaying various drought-related phenotypes, to comprehensively delineate components of the drought signaling pathway in Arabidopsis thaliana. Previously supported research has developed and refined robust quantitative proteomics technologies using isotope-assisted quantitation by mass spectrometry. These advanced methods will enable identification of groups of phosphoproteins that act in concert in response to important environmental changes, such as drought. The quantitative proteomic measurements will simultaneously provide insights into the response specific for water sensing as well as define a useful paradigm for applying this technology by members of the plant community. BROADER IMPACTS It is well recognized that drought represents the most severe limitation to providing an adequate supply of food for the world population. This project will identify key protein modifications that plants use to sense and respond to changes in water availability, and further develop and make available sophisticated mass spectrometry technology. In order to educate the public and our future leaders on why such expensive equipment and technology are required for research, the PI, Director of the UW Biotechnology Center, and an outreach staff have an established record of providing educational opportunities and activities for the community at large. In the current grant period, a new program will be developed aimed at the general area of "Measuring Molecules". The goal is to demystify mass spectrometers and demonstrate how they provide amazing sensitivity and power for detecting small amounts of good and bad compounds in our environment. The program will start with a discussion of the familiar uses for these instruments, such as at airports to screen for explosives, and on the planet Mars, for reporting on extraterrestrial molecules. The intention is to also develop and use classroom exercises at the high school and undergraduate level, for hands on experience with a new generation of mass spectrometer microscopes, including a MALDI-TOF/TOF that was obtained previously with an NSF MRI grant. Ultimately, through these activities there is hope to instill in the public a better appreciation for the theory and application of advanced technologies that are becoming ingrained in daily life.

智力优点随着无柄生物的植物已经发展了多种机制，以感知和反应许多不同的环境输入，包括非生物条件（例如光，水的可用性）和生物因素（例如昆虫，病原体）。该项目将通过定义将环境信号传输到植物细胞的特定分子步骤来提高我们对植物反应机制的理解。使用模型植物拟南芥和质谱中的高级技术（同位素辅助定量磷蛋白组学）该项目将识别通过磷酸化修饰的蛋白质，作为重要信号通路的一部分。最初的重点将放在ATHK1上，ATHK1是一种质膜组氨酸激酶，充当感测和应对植物细胞水的可用性（干旱）变化（干旱）的受体。该蛋白质是几十个组氨酸激酶的家族之一，对植物激素和环境参数的变化产生了短期和长期反应。组氨酸激酶至少部分地通过三种不同蛋白质中的组氨酸和天冬氨酸在组氨酸和天冬氨酸之间的一系列磷酸化作用。这些修饰（磷酸化）最终导致转录，代谢，细胞分裂和分化的大规模变化。除了组氨酸和天冬氨酸磷酸化外，干旱反应的遗传研究还暗示了钙依赖性的信号传导级联，其​​中包括丝氨酸/苏氨酸蛋白激酶。没有出现明确的模型来将这些化学不同的蛋白质磷酸化系统整合到对水利用率的总体响应中。在该项目中，将使用非放射性重型同位素和串联质谱法的植物代谢标记进行定量的磷光蛋白质组测量，以及在拟南芥中含有干旱信号通路的各种与干旱相关表型的突变体一起，展示了各种与干旱相关的表型的突变体。先前支持的研究已使用质谱法进行了同位素辅助定量，开发并完善了鲁棒的定量蛋白质组学技术。这些先进的方法将鉴定出响应重要环境变化（例如干旱）的磷蛋白组。定量蛋白质组学测量将同时提供对水感应特定的响应的见解，并定义了植物群落成员应用这项技术的有用范例。更广泛的影响众所周知，干旱代表了为世界人口提供足够的食物供应的最严重限制。该项目将确定植物用来感知和响应水可用性变化的关键蛋白质修饰，并进一步发展并提供复杂的质谱技术。为了教育公众和我们未来的领导者为什么需要研究这种昂贵的设备和技术，PI，UW Biotechnology Center的主任以及外展员工拥有为整个社区提供教育机会和活动的既定记录。在当前的赠款期间，将开发针对“测量分子”的一般领域的新计划。目的是揭开质谱仪的神秘面料，并演示它们如何提供惊人的灵敏度和力量来检测我们环境中少量的好和坏化合物。该计划将从讨论这些仪器的熟悉用途（例如在机场筛选炸药和火星地球上的机场）进行报告，以报告外星分子的报告。 目的是还要在高中和本科级别开发和使用课堂练习，以便拥有新一代质谱仪显微镜的经验，包括先前使用NSF MRI赠款获得的MALDI-TOF/TOF。最终，通过这些活动，希望在公众中灌输对日常生活中根深蒂固的先进技术的理论和应用更好的欣赏。